This application addresses broad Challenge Area (08) Genomics and specific Challenge Topic, 08-ES-104, Identification of alterations in epigenetic marks related to environmental exposures. In Avy mice that have an intracisternal A particle (IAP) in their agouti gene, exposure to a maternal diet-enriched in genistein or methyl- donors results in methylation of the IAP site and a graded shift from yellow coat color to pseudoagouti offspring. However, bisphenol A (BPA) exposure of pregnant black females that are carrying Avy fetuses results in more yellow coat color offspring. Yellow coat color offspring with a de-methylated promoter develop diabetes, obesity, and, in certain strains, cancer, but their darker siblings with a brown coat color remain healthy. These mice are a classic animal model to study epigenetics, as they are exquisitely sensitive to environmental changes. While one previous study has documented the overt coat color changes that occur in these mice in response to in utero exposure to BPA, which is an environmental estrogenic contaminant found in most plastic compounds, no study has examined the subtle phenotypic alterations, including in behavior and serum metabolite and hormone concentrations, and pathological changes that might also occur in the various coat color Avy mice. Moreover, in utero exposure of a single generation (F1) to BPA could result transgenerational effects into their great grand-offspring (F3), whose only exposure is through their predecessors. While the transgenerational effects of other compounds, including vinclozolin and diethylstilbestrol (DES) have been examined in rodents and humans (in the case of DES), not a single study to date has examined the transgenerational effects of BPA. Yet, such studies are highly relevant to humans, where we cannot easily assess the effects that exposure of one generation to endocrine disruptors, such as BPA, has on proceeding generations. However, with their 18 to 21-day gestation period and early sexual maturity (6 to 8wks of age), such transgenerational studies can readily be accomplished in mice. Herein, we will determine whether BPA can exert transgenerational effects by breeding black (a/a) females to viable yellow (Avy/a) males. Two weeks prior to breeding and throughout gestation, these females will be exposed to one of three doses of BPA (501/4g/kg/day, 5mg/kg/day, or 50mg/kg/day) through their diet. Control a/a females will be maintained on the AIN-93G diet. Pre- and post-weaning behavioral assessments will be performed on the pups to determine whether BPA affects their anxiety levels, learning and memory responses, and other developmental parameters. Femoral blood will be drawn weekly to measure the serum concentrations of metabolic and sex-regulating hormones, free fatty acids, and glucose, and their weight and overall appearance tracked. At 10 wks of age, the resultingY0 Avy males and females will be bred to control a/a females and a/a males, respectively, to determine whether any transgenerational effects can be passed through the male and female germlines. Additionally, their black coat color (a/a) (F1) siblings will be bred to determine if BPA underpins transgenerational effects independent of the Avy locus. The same experimental procedures will be repeated on the F2 and eventually the F3 generation, whose only exposure to BPA will be through their predecessors. These studies will permit us to examine the subtle changes in behavior, body weight, blood metabolites and hormone concentrations, and pathological changes that might ensue in viable yellow mice exposed in utero to BPA. Additionally, this study will examine the putative transgenerational effects that BPA can exert in a/a and Avy/a mice, the latter being a classic epigenetic animal model These studies have considerable relevance to humans, as they might serve as forewarning that our current exposure to "endocrine disruptors", such as BPA, has already compromised the welfare of our descendents. Should negative outcomes be detected in these transgenerational rodent studies, it might buttress public and congressional support to begin to curb BPA in the environmental and consumer goods to prevent such adverse effects in our descendents. Additionally, these data might provide insight into the impending medical conditions that our current exposure to BPA has imposed on our offspring, grand-offspring and even potentially great grand- offspring so that we are better prepared to therapeutically intervene in these generations. PUBLIC HEALTH RELEVANCE: Bisphenol A is one of the most commonly produced chemicals, as it used to make a variety of items, including plastics for baby bottles and re-usable water bottles, metallic cans, dental material, and in the production of cardboard and paper items. While numerous other studies have examined the effects that bisphenol A can exert on the individual, no previous study has ascertained the effects that it might have on the descendents of those subjected to this compounds. We will examine the behavioral, hormonal, and overall abnormal changes that might occur in in utero bisphenol A exposed mice and the ramifications that this early exposure has on their offspring through great grand-offspring to determine if bisphenol A exerts any potential transgenerational effects.